Rached E, Hooper N M, James P, Semenza G, Turner A J, Mantei N
Laboratorium für Biochemie II, Eidgenössischen Technischen Hochschule, ETH-Zentrum, Zurich, Switzerland.
Biochem J. 1990 Nov 1;271(3):755-60. doi: 10.1042/bj2710755.
Clones expressing renal dipeptidase (EC 3.4.13.11) have been isolated from a pig kidney cortex cDNA library after employing the polymerase chain reaction technique to amplify a region of the dipeptidase cDNA. The complete primary sequence of the enzyme has been deduced from a full length cDNA clone. This predicts a protein of 409 amino acids, a cleavable N-terminal signal sequence of 16 residues and two N-linked glycosylation sites. At the C-terminus of the predicted sequence is a stretch of mainly hydrophobic amino acids which is presumed to direct the attachment of the glycosyl-phosphatidylinositol membrane anchor. Expression of the mRNA for pig renal dipeptidase in Xenopus laevis oocytes led to the production of a disulphide-linked dimeric protein of subunit Mr 48,600 which was recognized by a polyclonal antiserum raised to renal dipeptidase purified from pig kidney cortex. Bacterial phosphatidylinositol-specific phospholipase C released renal dipeptidase from the surface of the oocytes and converted the amphipathic detergent-solubilized form of the dipeptidase to a hydrophilic form, indicating that Xenopus laevis oocytes can process expressed proteins to their glycosyl-phosphatidylinositol anchored form.
利用聚合酶链反应技术扩增猪肾皮质二肽酶cDNA的一个区域后，已从猪肾皮质cDNA文库中分离出表达肾二肽酶（EC 3.4.13.11）的克隆。该酶完整的一级序列已从一个全长cDNA克隆推导得出。这预测该蛋白有409个氨基酸，一个可切割的16个残基的N端信号序列和两个N-糖基化位点。在预测序列的C端是一段主要为疏水氨基酸的序列，推测它指导糖基磷脂酰肌醇膜锚定物的附着。猪肾二肽酶mRNA在非洲爪蟾卵母细胞中的表达导致产生了一种二硫键连接的二聚体蛋白，其亚基Mr为48,600，该蛋白可被用从猪肾皮质纯化的肾二肽酶制备的多克隆抗血清识别。细菌磷脂酰肌醇特异性磷脂酶C从卵母细胞表面释放出肾二肽酶，并将两亲性去污剂溶解形式的二肽酶转化为亲水形式，这表明非洲爪蟾卵母细胞可以将表达的蛋白加工成其糖基磷脂酰肌醇锚定形式。
